Published November 1, 1986
From the Division of Infectious Diseases, Department of Medicine, University of Virginia School
of Medicine, Charlottesville, Virginia 22908
This work was supported by Department of the Army grant DAMD 17-83-G-9565, and by NIH
grant AI 09504.
Abbreviations used in this ;taper: EF, edema factor ; FMLP, form yl-methionyl-leucyl-phenylalanine ; HBSS, Hanks' balanced salt solution ; LF, lethal factor ; MDP, muramyl dipeptide (N-acetylmuramyl-t.-alanyl-D-isoglutamine) ; PA, protective antigen; PMA, phorbol myristate acetate; PMN, polymorphonuclear neutrophils ; SOD, superoxide dismutase.
J. Exp. MED. © The Rockefeller University Press - 0022-1007/86/11/1700/10 $1 .00
Volume 164 November 1986
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Anthrax toxin, a critical virulence factor of Bacillus anthracis (1-3), consists of
three protein components : protective antigen (PA)', edema factor (EF), and
lethal factor (LF) . PA, the major antigen of acquired immunity (4, 5) evidently
combines with susceptible cells, forming a receptor for EF and LF (6). EF, initially
recognized by its ability to produce edema in tissues, has been identified as an
adenylate cyclase, which, in combination with PA, forms adenosine 3',5'-monophosphate (CAMP) in susceptible cells; EF alone forms cAMP in a reconstituted
cell-free system (6, 7). LF is identified by its acute lethality in animals when
injected in combination with PA; its mode of action is unknown despite extensive
studies (4, 8, 9). None of the factors by itself produces acute toxic reactions.
Keppie et al . (10) presented evidence that the toxin was antiphagocytic and
antibactericidal by virtue of an action on phagocytic cells. Despite the significance
of these effects for the further understanding of pathogenesis, only recently have
efforts been made to elucidate their mechanism.
By analogy with other bacterial adenylate cyclases, we anticipated that PA plus
EF would inhibit chemotaxis of PMN; we found instead that pretreatment with
PA plus EF or PA plus LF markedly stimulated chemotaxis (11). It seemed
possible that the observed stimulation was associated with inhibition of the normal
modulation of chemotaxis by oxidative or other secretory products of stimulated
PMN (12, 13). PA plus EF, but not PA plus LF, was reported (14) to inhibit
phagocytosis of opsonized B. anthracis by human polymorphonuclear neutrophils
(PMN) and to block chemiluminescence induced by opsonized B. anthracis or by
phorbol myristate acetate (PMA) .
Pretreatment with toxin reduced the release of superoxide anion (C)-after
PMN were stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). Initially, the inhibition of 02 release was variable, and it was suggested that
spontaneous fluctuations in the level of contamination with pyrogen were a
contributory factor . A conceptual basis for the variability was provided by reports
(15, 16) that PMN isolated under conditions that minimize exposure to bacterial
lipopolysaccharide (LPS) produced weak oxidative responses to FMLP and
Published November 1, 1986
170 1
certain other stimuli . Preincubation with LPS at concentrations as low as 1 ng/ml
produced a severalfold increase in 02 release, an effect referred to as priming.
The marked increase in the release of 02 and lysosomal enzymes associated with
priming increases not only the antimicrobial potential of PMN but also their
ability to damage adjacent tissue (17).
We found that unprimed PMN treated with PA plus EF or PA plus LF resist
subsequent priming by LPS and priming by N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]), a synthetic glycopeptide mitogen analogous
to bacterial peptidoglycans (18). The inhibition of priming results in marked
inhibition of 02 release relative to primed controls after stimulation with FMLP,
but not after stimulation with PMA . The inhibition of priming represents a
mechanism, not described previously, whereby a bacterial toxin dampens the
oxidative response of PMN .
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Materials and Methods
Preparation of PMN. Human blood was drawn into 1/10 vol of 3 .8% sodium citrate,
prepared by dilution of 46.7% sodium citrate solution (Alpha Medical Products, Providence, RI) with water for injection . PMN were isolated by dextran sedimentation and
hypotonic lysis of erythrocytes and washed twice according to the method of Guthrie et
al. (15), except that Hanks' balanced salt solution (HBSS) without phenol red (Whittaker
M .A. Bioproducts, Walkersville, MD) was used for washing and final suspension of PMN .
In exploratory experiments, PMN gave variable production of 02 after stimulation; this
effect was associated with clumping during preincubation and was ascribed to the presence
of Ca" and Mg` in the HBSS and the variable carry-over of citrate . The variability and
clumping were overcome by addition of 2 mM sodium citrate to the HBSS used for
washing and suspension of PMN ; the final concentration in the test was 1 mM. The
suspensions were >>-80% PMN; they were diluted to 8-10 X 106 cells/ml (total count) .
Great care was taken to avoid the uncontrolled introduction of pyrogens by use of
pyrogen-free, single-use plastic- or glassware and pyrogen-free solutions, which were
stored at -70 °C when possible or handled under aseptic conditions and refrigerated. .
Anthrax Toxin . PA, EF, and LF were supplied by Dr. S. H. Leppla of the U .S. Army
Medical Research Institute of Infectious Diseases, Frederick, MD. They resembled previous preparations in purity and specific activity (6, 19). The stock solutions were diluted
in HBSS to 30 jAg/ml, dispensed in amounts sufficient for one experiment, and held at
-70 0C.
Toxin Treatment, Priming, and Stimulation of PMN. PMN were exposed to the toxin
components, alone and in various combinations, for 1 h at 37 ° C, followed by addition of
the priming substance and incubation for an additional hour at 37 ° C. Cytochrome c and
stimulant were then added, and the tests were incubated for 10 min at 37'C in a shaker
water bath, cooled, and centrifuged for 15 min at 3,000 g. Each determination consisted
of two tubes, which were identical except that one tube received superoxide dismutase
(SOD) and was placed in ice immediately before the addition of cytochrome c and
stimulant . The release of 02 in the incubated tube was determined from the difference
in absorption of supernatants of each pair of tubes at 550 nm.
Tests in a final volume of 600,ul were set up in duplicate in 12- X 75-mm plastic tubes
(2054 ; Falcon Labware, Becton, Dickinson & Co., Oxnard, CA) . The final concentrations
were: PA, EF, LF, as indicated; human serum albumin, 0.2% ; PMN, 4-5 X 106/ml ; LPS
and MDP, as indicated; SOD (when present), 0 .1 mg/ml ; cytochrome c, 0.12 mM;
stimulant, 10-7 M (FMLP) or as indicated (PMA) . Concentrations of toxin and primer are
referred to the final 600-,al volume. SOD from bovine erythrocytes, cytochrome c type
VI, and FMLP were obtained from Sigma Chemical Co., St. Louis, MO; PMA was from
Consolidated Midland Corp., Brewster, N.Y . FMLP and PMA were dissolved in dimethyl
sulfoxide at concentrations of 0 .01 M and 100 and 1 mg/ml, respectively, held at -70°C
Published November 1, 1986
170 2
in small quantities, and thawed and diluted in HBSS immediately before use . LPS from
Escherichia coli K235 (List Biological Laboratories, Campbell, CA) was suspended in HBSS
at 1 mg/ml, dispersed by brief sonication, and stored at 4°C . MDP (Sigma Chemical Co.)
was dissolved in HBSS, held in small quantities at -70°C, and thawed and diluted
immediately before use. Human serum albumin, in 5% solution for clinical use, was
obtained from Cutter Laboratories, Cutter Biological, Berkeley, CA, or the New York
Blood Center. The undiluted solution gave positive tests for LPS by the Limulus amebocyte
lystate test (using Pyrotell reagent ; sensitivity, 0.006 ng of LPS ; obtained from Associates
of Cape Cod, Inc., Woods Hole, MA), but gave negative tests at a 1 :5 dilution . There was
no indication that either preparation caused priming of PMN at the 0.2% final concentration used.
Determination of02 Release . The difference between each pair ofabsorption measurements was divided by the PMN count in millions and the extinction coefficient of 1 .85 X
104 cm2 mmol- ' (20). This yielded 02 released in millimoles per 106 PMN. Control tests
without anthrax toxin were set up in duplicate for each set of conditions, so that the
effects of toxin could be determined . The patterns of inhibition of 02 release were
consistent in repeat experiments using PMN from different donors, but the levels of 02
varied somewhat, which presumably reflects individual differences in the proportion of
PMN that responded to FMLP (21). Accordingly, the percent changes in 02 release
relative to the mean control value without toxin were determined, means and standard
deviations were calculated for replicate experiments, and these values were reconverted
to 02 in nanomoles per 106 PMN by reference to the respective mean control values.
The means and standard errors are represented in the figures ; the values of n, the number
of replicate experiments, are given in the legends .
Effects of LPS Priming and Anthrax Toxin Treatment on 02 Release . Human
PMN isolated with minimal exposure to bacterial products released relatively
small amounts of 02 upon stimulation with FMLP; treatment with a range of
concentrations of LPS for 1 h at 37'C (priming) increased their subsequent
release as much as eightfold (Fig. 1). Smaller but appreciable effects were
observed with concentrations of LPS as low as 1 ng/ml . The levels of response
of control PMN to low concentrations of LPS varied somewhat among cells from
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Release of 02 after stimulation of PMN with 10' M FMLP as a function of
concentration of LPS during priming. PMN were incubated for 1 h at 37°C with control
buffer, or PA plus EF (each 0.25 Wg/ml), or 0.25 kg/ml PA plus `?.5,ag/ml LF. LPS was added
and tests were incubated for 1 h at 37°C and stimulated with FMLP, and the 02 released in
10 min was measured. n = 4.
Published November 1, 1986
different donors, which perhaps reflects a variation in humoral immunity to LPS
among individuals (22).
Pretreatment of PMN with PA plus EF for 1 h at 37 ° C markedly reduced the
levels of 02 released after LPS priming; reduction was ?90% in the range from
1 to 100 ng/ml LPS . The pretreatment produced consistent reductions in the
small amount of 02 released from PMN not primed with added LPS. Pretreatment with PA plus LF also reduced the release of 02 after priming and
stimulation, but the effect was less marked than the effect of PA plus EF,
especially at higher concentrations of LPS. After priming with 1 ng/ml of LPS,
however, the inhibition of 02 release was 84% .
To explore the interactions of priming and anthrax toxin treatment of PMN,
experiments were set up with PA plus a range of concentrations of EF or LF, in
which 02 release was compared without priming, with priming with LPS after
exposure to toxin, and with priming before exposure to toxin (Fig. 2). PA, when
present, was held constant at 0 .25 ug/ml . It is evident that both PA plus EF and
PA plus LF produced strong inhibition over a range of concentrations when LPS
priming was carried out after exposure to anthrax toxin; reversing this order
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2. Release of 02 after stimulation of PMN with 10 -7 M FMLP as a function of
concentrations of PA, EF, and LF present during preincubation, and of conditions of priming
with 3 ng/ml LPS. In A, PMN were exposed to PA, EF, or LF in the concentrations shown
for 1 h at 37°C, after which LPS was added and the tests were incubated for 1 h at 37°C .
The release of 02 in 10 min was determined after stimulation with FMLP. Dose-related
inhibition of 02 release relative to the control without anthrax toxin is evident with both PA
plus EF and PA plus LF . In B, tests were carried out in the same manner, except that HBSS
was added instead of LPS. In the absence of priming, the small amount of 02 released in the
control without anthrax toxin makes it difficult to detect inhibition by PA plus EF or PA plus
LF . In C, PMN were exposed to LPS before exposure to anthrax toxin; otherwise, conditions
were the same as in A. Inhibition by PA plus EF and PA plus LF relative to the control is
much smaller than in A. n = 4-6. Tests in which PA, EF, or LF were added singly gave results
essentially identical to controls without toxin, and have been omitted.
Published November 1, 1986
1 104
4. Release of 02 after stimulation of PMN with 10 -7 M FMLP as a function of
concentration of PA, EF, and LF present during preincubation, and of priming with 100
ng/ml MDP. The experimental design was otherwise the same as for Fig. 2 . n = 6-10 .
markedly reduced the inhibitory effect . Strong inhibition was obtained with 0 .01
Ag/ml of EF; LF was less active, but produced 84% inhibition of 02 release at
0.5 Ag/ml . Without priming, toxin treatment produced slight inhibition of 02
release relative to the low value of the control without toxin.
Effects of Priming with MDP and Treatment with Anthrax Toxin on 02 Release . Experiments similar to those described above were carried out, except
that MDP was used instead of LPS (Figs. 3 and 4). The priming effects were
slightly smaller than with LPS, and the higher concentrations of MDP did not
overcome the inhibitory effects of PA plus LF to as great a degree as did LPS.
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3 . Release of 02 after stimulation of PMN with 10 -7 M FMLP as a function of
concentration of MDP during priming. PMN were incubated for 1 h at 37°C with control
buffer, or 0.25,ug/ml PA plus 0.5 ag/ml EF, or 0.25 Pg/ml PA plus 0.5 wg/ml LF . MDP was
added to reach the concentrations shown, and tests were incubated for 1 h at 37 °C. PMN
were stimulated with FMLP and the 02 released in 10 min was measured . n = 8.
Published November 1, 1986
In addition, PA plus EF added after priming resulted in somewhat greater
inhibition of 02 release than was observed with LPS priming. The overall effects
were similar.
Comparison of FMLP and PMA Stimulation after Anthrax Toxin and MDP
Priming. Five conditions of stimulation were compared : no stimulus, 10-7 M
FMLP, and three concentrations of PMA: 20, 5, and 2 ng/ml . Four combinations
of anthrax toxin treatment and MDP priming were investigated for each condition of stimulation: no anthrax toxin and no priming, no toxin and MDP priming,
PA plus EF and MDP priming, PA plus LF and MDP priming. The results (Fig.
5) are consistent with those reported above for FMLP stimulation and MDP
priming. In contrast, with PMA there was no evidence of priming by MDP, and
no inhibition of 02 release by PA plus EF or PA plus LF.
The marked inhibition of FMLP-induced 02 release produced by pretreatment of PMN with PA plus EF or PA plus LF before priming with LPS or MDP
indicates that anthrax toxin alters the cells in such a manner that they resist
priming . When PMN were primed first and then exposed to the toxin, only
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Comparison of the effects of stimulation of PMN by FMLP and three concentrations of PMA on priming effects of 100 ng/ml MDP, and on the inhibitory effects of PA plus
EF and PA plus LF . PMN were exposed first to anthrax toxin components or control buffer
for 1 h at 37°C, after which MDP or control buffer was added to a final concentration of 100
ng/ml and incubation continued for an additional hour at 37°C . The release of 02 in 10 min
was measured after no stimulation, after stimulation with 10' M FMLP, or after stimulation
with PMA at 20, 5, or 2 ng/ml. n = 4-6.
Published November 1, 1986
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slight inhibition was observed . These effects indicate that inhibition by toxin and
priming, once established, are not readily reversed . The concept of inhibition of
priming by toxin is compatible with the critical role of toxin in virulence (2, 3,
10) and the major increase in the potential to release 02 and lysosomal enzymes
associated with priming (15-17) . The release of these substances by PMN
represents a major mechanism of bactericidal activity (23, 24); the possession of
mechanisms for inhibiting bactericidal effects would contribute significantly to
The results in Fig. 2 provide explanations for the initial difficulties in obtaining
consistent effects of anthrax toxin when LPS was not controlled, and may explain,
in part, the results of others (14) . Exposure of PMN to 3 ng/ml LPS, a
concentration readily obtained in solutions not carefully handled to exclude
pyrogens, initiated a level of priming that was not inhibited appreciably by
subsequent treatment with anthrax toxin. In other experiments, the exclusion
of LPS prevented priming and reduced the release of 02 in controls to a level
at which inhibition by treatment with the toxin was difficult to detect . Only when
exposure to LPS was controlled with respect to concentration and timing were
clearly recognizable and consistent effects of anthrax toxin obtained .
A model system that includes priming by LPS does not reproduce fully the
processes occurring during anthrax because B. anthracis does not produce LPS.
The observation that MDP is also active in priming PMN, a possibility raised
initially by analogy with activation of macrophages (25, 26), provides a closer
link to B. anthracis because MDP is related to peptidoglycans of bacterial cell
walls in structure and activity (26, 27). MDP has been reported to be inactive in
priming of PMN under other conditions, however (28) .
The priming of PMN by LPS not only increases the release of 02 in response
to stimuli, but also enhances the release of lysosomal enzymes, induces spontaneous changes in shape (16), and modulates chemotactic responsiveness (16, 29).
Our previous observation (11) that chemotaxis was stimulated by treatment with
anthrax toxin can be explained by the assumption that control PMN were primed
by LPS introduced during their isolation, resulting in reduced chemotactic
responsiveness . This priming was inhibited in the presence of the toxin, producing apparent stimulation of chemotaxis . This concept appears more probable
than the tentative explanation suggested previously, that anthrax toxin inhibits
the secretory activities of PMN that modulate chemotaxis .
Pretreatment with amounts of anthrax toxin that produced almost complete
inhibition of 02 release after stimulation with FMLP had no effect on 02 release
after stimulation with PMA. PMA stimulation also did not reveal evidence of
priming as a result of pretreatment with MDP or (not shown) LPS ; this absence
of inhibition by the toxin when priming does not occur provides additional
evidence that the toxin acts to prevent priming. Guthrie et al . (15) obtained
priming by LPS with PMA stimulation, but the ratio of 02 with LPS treatment
to 02 without LPS was 1 .64 for PMA, compared with 7.76 for FMLP . Our
results with PMA suggest that priming alters the polyphosphoinositide transmembrane signal mechanism at a point proximal to activation of protein kinase C,
since PMA activates protein kinase C directly (30) .
Published November 1, 1986
170 7
The authors thank Gail Sullivan for advice on the selection of methods and interpretation
of results, and Agbor Egbewatt and Craig Lombard for assistance.
Received for publication 15 May 1986 and in revised form 30 June 1986 .
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We studied the pretreatment ofhuman polymorphonuclear neutrophils (PMN)
with purified preparations of the anthrax toxin components-protective antigen
(PA), edema factor (EF), and lethal factor (LF)-and their effects on release of
superoxide anion (02) after stimulation with the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) . PMN isolated in the absence of lipopolysaccharide (LPS) (<O.1 ng/ml) released only small amounts of 02 after FMLP
stimulation ; pretreatment with anthrax toxin had little effect . The release of
02 was increased fivefold by prior treatment with 3 ng/ml LPS for 1 h at 37 ° C,
an effect referred to as priming. PMN were primed to an equivalent extent by
treatment with 100 ng/ml N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl
dipeptide [MDP]) . Pretreatment of PMN with anthrax toxin components PA
plus EF or PA plus LF inhibited priming by LPS or MDP, as shown by the
reduction in the release of 02 up to 90% relative to controls not treated with
toxin ; single toxin components were inactive . The inhibition was markedly
reduced when priming with LPS or MDP was carried out before exposure to
toxin . 02 release after stimulation by phorbol myristate acetate was not increased
by priming, and pretreatment with toxin did not inhibit 02 release after this
stimulus. Evidently, anthrax toxin inhibits the priming that is normally induced
in PMN by bacterial products and is necessary for the full expression o£ antimicrobial effects .
Published November 1, 1986
170 8
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